This competing continuation is a proposal for ultrastructural and functional studies of lens fiber cell junctions. The two major types of junctions are gap junctions (GJs) which mediate the intercellular transport of ions and small molecules and square array junctions (SAJs) which are formed by the close association of undulating membrane pairs in the tongue-and-groove interdigitations. One suggested role for SAJs is to limit the low refractive index extracellular space compartment. During the first period of support, it was firmly established that SAJs show distinctive and pronounced modifications during aging in normal bovine lenses and nuclear human senile cataracts. The results suggest that SAJs play a key role in the formation of nuclear opacities. The amount of SAJ also increased significantly in the early stages of cataract formation in diabetic rabbit lenses. We plan to test the hypothesis that the fiber cell membrane is the initial site of damage in the formation of many cataracts by examining the ultrastructure of junctional specializations in fiber cell membranes. This proposal is for the study of bovine and human lenses primarily employing thin-section and freeze-fracture-etch electron microscopy to further characterize the aging process and the formation of scattering centers. The thin-section images will be derived from thick Vibratome tissue slices of whole lenses using methods refined during the initial grant period. These slices permit excellent preservation throughout the lens and facilitate comparisons of transparent and opaque regions. The freeze-fracture-etch technique will give the distribution of membrane proteins which is valuable for identifying junctions and their alterations with age. New studies will be initiated on human diabetic lenses, especially those with posterior subcapsular cataract because this cataract contains extensive aggregations of SAJs. New experiments are also proposed to investigate the intercellular movement of fluorescent dyes using the laser scanning confocal microscope. The confocal microscope is ideal for focusing on selected intact fiber cells within thick Vibratome sections without interference from other cells of the cut surfaces. The transport of dye through GJs is important for understanding why GJs in the interior of the lens have different channel properties than GJs in the periphery or in other tissues. We will also evaluate the hypothesis that SAJs offer an alternate route for intercellular transport through channels of MIP26, the major membrane protein that forms the square arrays. Water and ions could pass through MIP26 channels in one square array into the extracellular space then into adjacent fiber cells through other MIP26 channels. continued structural studies of SAJs in isolated bovine membranes are proposed employing high resolution tantalum replicas to determine the size of single MIP26 subunits and to characterize the channels in large crystalline arrays. The separation of the membranes in SAJs will be investigated as a function of ionic strength and the concentration of divalent cations in order to determine the mechanism of adhesion. These studies will provide important new information concerning the role of intercellular junctions in aging and about the general mechanisms underlying cataract formation in humans.